Identification Of The Causal Agent Of Leaf Blight On Blueberry And ISSR Fingerprinting Of Botrytis Cinerea Isolates

Blueberry is a newly developing fruit tree with high value in recent years. Since it was introduced to China in the 1980s, the cultivated areas were enlarged from small planted area to large-scale commercial cultivation and have been expanded to more than 10 provinces in China.With blueberry cultivating areas enlarging, market expanding, and the demand for blueberry increasing, various pests were emerged and how to protect plants from them has become a critical matter. In our labs, we mainly focus on the further reseach and identification of new diseases of blueberry. In 2014, a new disease was found in the investigation, and the task group was listed as the key identification object because this disease was easily confused with gray mould. Meanwhile, it was one of the main diseases in blueberry cultivation because of the large damage caused by gray mold in the production. The samples of blueberry gray mould disease used in this study were collected between 2012 to 2015. The pathogens were sequentially isolated, purified, and stored. The species identification, ISSR fingerprint, fungal diversity, pathogenicity and resistance were studied and the progresses are as follows:1.a new leaf blight was found in blueberry cultivar area in Jiangxi province. It is easily confused with gray mould caused by Botrytis cinerea, early it starts to infect from leaf margin or tip, the leaf spot belongs to irregular lesion and late dry in a curly shape. It is seriously affected plant growth, resulting in great economic losses. After pathogenicity testing, morphology identification and its TEF-1a sequences, the pathogen is identified as Fusarium asiaticum(Fusarium asiaticum, FA for short), which is new record species of pathogen on blueberry2. Respectively a total of 273 strains were obtained from host such as blueberry, tomato, cucumber, eggplant, etc. belonging to Botrytis.35 representative strains were screened out as the PDA culture traits and morphological differences for further study. The morphological identification based on ITS and BC729 sequences proved the population for the first time; and then the G3PDH, HSP60 and RPB2 gene system combined with phylogenetic analysis was used for second validation. The results showed that all 35 representative strains belong to Botrytis cinerea.3. Although selected 35 representative strains are in the same population, obvious differences were found in the PDA colony in spore production and sclerotium characteristics, then the genetic fingerprints were studied. Nine typical fingerprints of B. cinerea based on 3 ISSR primers were showed and the result was verified. Three different primers, named 807, 864 and 886, were screened out for the construction of B.cinerea genetic fingerprints with stability repeatability, obvious main bands and a wide spacing. The optimal annealing temperatures of the 3 primers were 51℃,52℃ and 49℃ respectively. All 35 representative strains were amplified by primer 807 with 3 different types of fingerprints detected (as a,b and c), primer 864 with 4 different types of fingerprints (as Ⅰ,Ⅱ,Ⅲ and Ⅳ), and primer 886 with 5 different types (as A,B,C,D and E). The results of the above three primers were analyzed and combined into 9 types (N1,N2,N3,C1,C2,C3,C4 C5 and S1). The 3 primers were used to construct the DNA fingerprints of 35 B. cinerea strains,32 strains were used to verify the ISSR results obtained. Randomly selected from the other strains of the representative strains of 32 strains of B. cinerea for fingerprints verification.The results showed that 32 strains belonged to N1, C2, C3, C4, C5, S1, and so on. The results showed that there was no new type.4. There was no significant correlation between the experimental analysis of the relationship between the ISSR fingerprints and the pathogenicity and resistance.67 strains having finished the pathogenicity of the ISSR fingerprint analysis were measured. The results of the study for C1 type of strong, weak pathogen each accounted for 50%; pathogenicity of C2 type of strong and weak pathogenicity, avirulent strains accounted for 23.3%,30.1% in pathogenic strains; C4 type in grade 2, grade 1,0 pathogenic strains,15% each, grade 3 pathogenic strains accounted for 65%; similarly, other types of fingerprints also appeared similar, i.e. the patterns under different virulence levels strains were distributed, the existence of the phenomenon of pathogenicity differentiation. After comparison, four kinds of fungicides were screened on Botrytis cinerea isolates were resistance determination: carbendazim, procymidone, pyrimethanil, fludioxonil. The MIC (minimum inhibitory concentration method) method as a method of resistance identification. C1, only one of the representative strains of 35 strains of different types of primary resistance identification. The results of 35 botrytis cinerea strains, the CPC has been Pythium strains resistant to 21 strains, carbendazim resistant strains 34 strains, its mildew amine resistant strains (26 strains, Luo Bromothalonil resistant strains strains. The Pythium resistance screening test results, type C2 type N3 of all strains were resistant strains, Nl, N2 and S1 flora only a strain of representative strains were sensitive to; 35 strains only a strain belongs to C2 group strains and drug resistance, and the remaining 34 strains showed sensitive to carbendazim resistance test, in addition to the flora in a strain resistant strains and other strains representing all of the performance for the resistance.